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Balanites roxburghii is an underutilized species native to India and known for its medicinal and nutritional properties. Seeds possess a high amount of oil, which could be a new source of edible oil. Here we report the compositional analysis of B. roxburghii seed oil (BRSO) and its variability among the South Indian populations. Oil content ranged from 43.2 % to 61.0 %. Oleic, linoleic, palmitic, and stearic were the major fatty acids, and their content varied greatly among the accessions. Carotenoid content was up to 22.6 mg/kg oil. α-Tocopherol ranged between 1.21 and 10.6 mg/100 g, whereas β and γ-tocopherols ranged between 0.89 and 4.98 mg/100 g. β-Sitosterol was the major phytosterol (52.5–147 mg/100 g), and squalene quantity was significant (up to 65.4 mg/100 g). BRSO has no cytotoxic effects on 3T3-L1 cell lines. Results suggest that the BRSO is a significant source of oil and nutraceuticals.